Anti-dementia substance from hericium erinaceum and method of extraction

ABSTRACT

A fat-soluble fraction extracted from the fruiting body of  Hericium erinaceum  is demonstrated to inhibit the neuronal toxicity of amyloid beta-peptide (Aβ) and induce the synthesis of nerve growth factor (NGF), and has great potential as an active ingredient for pharmaceutical products, health food products, food products and/or beverages to prevent and/or treat dementia, especially Alzheimer-type dementia. This invention is to provide the bioactive fraction and its preparation method.

FIELD OF INVENTION

The present invention relates to the field of bioactive substancesderived from natural products. More specifically, the present inventionrelates to a fraction with anti-dementia effects, especiallyanti-Alzheimer dementia effects, extracted from the fruiting body of themushroom Hericium erinaceum, and a method for preparing the fraction.

BACKGROUND OF THE INVENTION

Recently, the number of patients with Alzheimer type dementia andcerebrovascular dementia is increasing at a remarkable rate in thosecountries with a rapidly aging population and this has become a serioussocial problem. Among these dementias, Alzheimer type dementia occursdue to neurodegeneration. It is symptom-free in its early developmentperiod. Once a patient is diagnosed with advanced stages the symptomsare irreversible and incurable with current medical technology.Therefore, a need exists for a bioactive substance that helps to reducethe risk of neurodegeneration and/or to delay the resultant symptoms.

Research is focusing on the clarification of the mechanism under whichAlzheimer type dementia progresses and the development of new drugs totreat the condition. There have been a number of reports published. Ithas been found that patients with Alzheimer type dementia have similarbrain abnormalities as those patients with basal forebrain cholinergicneuron syndrome (BFCN) which is also associated with loss of memory andlearning capabilities due to neurodegeneration, neuronal atrophy, andneuronal loss. Because nerve growth factor (NGF) exerts a trophic actionon BFCN, lack of NGF is considered to be one of the causes of thesedementias. However, NGF is a protein, and may be degraded in the bodyand/or cannot pass through the blood-brain barrier when administratedorally. Therefore, it has become an objective of medical andpharmaceutical researchers in this area to discover or developsubstances with low molecular weight which can pass through blood-brainbarrier and induce the synthesis of NGF in the brain.

In addition, it has been reported that amyloid beta-peptide (Aβ) is oneof the inducers of Alzheimer type dementia since it causes theinflammation and neurodegradation inside the neuron associated withAlzheimer's and leads to death as it accumulates in the brain.Therefore, the inhibition of the toxicities of amyloid beta-peptide isconsidered to be one of desired ways to prevent and treat the Alzheimertype dementia.

Some investigators have studied the inductive effects of NGF synthesisof H. erinaceum extracts. (See, for example, Kawagishi, et al.Tetrahedron Letters, 32 (35):4561-4564 (1991); Kawagishi, et al.,Phytochemistry, 32(1):175-178 (1993); Japanese Patents Nos. 04-266848and 04-275285.) Another investigators have reported the inhibitoryeffects of Aβ toxicity of different H. erinaceum extracts (See, Nagai,et al. J. Nutritional Chemistry, 17:525-530 (2006); Japanese Patent No.3943399). The cited references all disclose extraction methods thatutilize toxic organic solvents in the extraction procedures renderingthem problematic for use in industrial or large scale extractionprocedures designed to produce compounds for clinical use. In addition,the results disclosed in the references cited above are confined to invitro experiments.

In this regard, because neurodegenerative diseases typically Alzheimertype dementia have a long incubation period prior to diagnosis, reducingthe risk before the identification of visible symptoms of the disease isimportant. It would be advantageous to be able to reduce the risk ofneurodegeneration by the consumption of food products if available andan important of option for target populations. Therefore, there is aneed in the field for a method for extracting compounds from H.erinaceum using safe chemicals and shown to be effective in vivo.

SUMMARY OF THE INVENTION

The present invention broadly comprises a method for preparing afat-soluble fraction from H. erinaceum by the steps of extracting thefruiting body of H. erinaceum with 95% ethanol at room temperature,obtaining the ethanol extract by filtration or centrifugation,concentrating the ethanol extract by removing ethanol under reducedpressure, adding 4˜8 volumes of water into the concentrated extract,collecting the resulting floating matter on the surface of theconcentrated extract after standing at 4˜10 degrees C. for 8˜12 hours;and, drying the collected floating matter. The invention also includes afat-soluble fraction of H. erinaceum comprising fat-soluble componentsthat include benzyl alcohol derivatives, chromane derivatives, andphosphatidylethanolamine derivatives with anti-dementia and/oranti-Alzheimer type dementia activities.

In another embodiment, the present invention broadly comprises abioactive fraction of fat-soluble components of H. erinaceum produced bythe steps of extracting the fruiting body of H. erinaceum with 95%ethanol at room temperature, and obtaining ethanol extract by filtrationor centrifugation, concentrating the ethanol extract by removing ethanolunder reduced pressure, adding 4˜8 volumes of water into theconcentrated extract, collecting the resulting floating matter on thesurface of the concentrated extract after standing at 4˜10° C. for 8˜12hours; and drying the collected floating matter. The bioactive fractionmay be incorporated into pharmaceutical products, health food products,food products, and/or beverages. A bioactive fraction is defined as thefat-soluble components of H. erinaceum that include anti-dementia and/oranti-Alzheimer's compounds, and found to reduce or eliminate one or moresymptoms of dementia and/or Alzheimer's disease.

An object of the invention is to provide a bioactive fraction composedof fat-soluble components that include benzyl alcohol derivatives,chromane derivatives, and phosphatidylethanolamine derivatives as mainbioactive compounds.

A second object of the invention is to provide a bioactive fractionhaving anti-dementia activities, especially anti-Alzheimer type dementiaactivities, as seen by increased the synthesis of NGF and the reductionor elimination of the toxicity of amyloid beta-peptide.

A further object of the invention is to provide a process of extractingthe bioactive fraction that has a high measure of safety.

An additional object of the invention is to provide products thatinclude the bioactive fraction of H. erinaceum.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

The nature and mode of operation of the present invention will now bemore fully described in the following detailed description of theinvention taken with the accompanying drawing figures, in which:

FIG. 1 is a flow diagram depicting the method used to extract the fatsoluble fraction from H. erinaceum;

FIG. 2 is a flow diagram depicting the method used to fractionate thefat soluble fraction obtained from H. erinaceum; and,

FIG. 3 is a graph showing the effects on cell viability afteradministration of increasing doses of HEF.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION

While the present invention has been described with respect to what ispresently considered to be the preferred embodiments, it is understoodthat the invention is not limited to the disclosed embodiments. Thepresent invention is intended to cover various modifications andequivalent arrangements included within the spirit and scope of theappended claims.

The bioactive fraction of the present invention is obtained by theextraction and fractionation procedures described in Example 1 below inwhich ground or pulverized fruiting body of the mushroom H. erinaceum isextracted with ethanol. The obtained ethanol extract is concentrated byremoving ethanol under reduced pressure, and then water is added. Theresulting floating matter is collected after standing at 4˜10° C. for8˜10 hours. The floating matter is dried to obtain a fraction describedin Example 2 below.

Example 1

The method of obtaining the bioactive fraction of the present inventionis described as follows:

As seen in the flow diagram in FIG. 1, 1,000 g of the dried fruitingbody of Hericium erinaceum was extracted with 10 L of 95% ethanol atroom temperature for 2˜3 hours, and the ethanol extract was obtained byfiltration or by centrifugation. The obtained ethanol extract wasconcentrated by removing ethanol under reduced pressure, and then 4˜8volumes of water were added into the concentrated ethanol extract. Thefat-soluble components separated out from the solution by forming alayer of floating matter on the surface of the solution after standingat 4˜10° C. for 8˜10 hours. The resulting floating matter was collectedby a skimming system or by a pipetting system. The floating matter wasdried to yield approximately 19.5 g (dried weight) of brown substance(named as HEF).

Example 2

The fraction of the invention (the floating matter) obtained above inExample 1 was analyzed to identify its characteristics.

The fraction is soluble in methanol or ethanol, insoluble in water, andis thermostable.

The fraction was completely dissolved in 95% ethanol, and chloroform wasadded into the residual solution after ethanol was removed under reducedpressure. The chloroform-soluble components were obtained by solventpartition between chloroform and water.

As seen in FIG. 2, the chloroform-soluble components were fractionatedand purified by high performance liquid chromatography (HPLC) on varioussilica gel columns. The purified compounds were determined using FastAtom Bombardment Mass Spectrometry (FAB-MS), IR spectrum (IR) andNuclear Magnetic Resonance (NMR) methods well known to those havingskill in the art, and identified as benzyl alcohol derivatives, chromanederivatives, and phosphatidylethanolamine derivatives, respectively.

The following examples describe several experiments performed in orderto investigate the biological activities of the substance of thisinvention (HEF).

Example 3

Six groups of Sprague-Dawley male rats approximately 11 weeks old weredivided into control, amyloid beta-peptide (Aβ), Aβ+Donepezil (Aβ+D),Aβ+HEF-H (high dose), Aβ+HEF-M (middle dose), and Aβ+HEF-L (low dose)groups. The rats of the Aβ, Aβ+D, Aβ+HEF-H, Aβ+HEF-M, and Aβ+HEF-Lgroups were separately injected with 5 μL of amyloid beta-peptide(Aβ₁₋₄₀) solution at a concentration of 2 μg/μL into both sides of brainhippocampus for preparing the rats with Alzheimer type dementia (ADrats). The control group was injected with 5 μL of saline. From thefourth day after the injection, the Aβ+D group was administered with theanti-Alzheimer's drug Donepezil (brand name: Aricept) at a dose of 1mg/kg (equal to 2 times of the recommended therapeutic dose clinically),and Aβ+HEF-H, Aβ+HEF-M, and Aβ+HEF-L groups with HEF separately at thedoses of 24 mg/kg, 12 mg/kg, and 6 mg/kg, and the control and Aβ groupswith same volume of saline by stomach tube once per day for four weeks.From fourth week after the administration, learning and memory-relatedbehavior was assessed using the Morris Water Maze Test on ten ratsarbitrarily-chosen from each group once a day for one week (from day 1to day 6 the time to arrive at the platform is measured, and on day 7the platform is removed and the frequency to cross (pass by) theplatform location is counted). After completing the behavioral test, therats were sacrificed. The cerebral tissues separated from half of therats of each group were used for pathological examination, and theolfactory bulb, hippocampus, locus ceruleus, and cerebral cortex fromanother half were used to determine the NGF content with enzyme-linkedimmunosorbent assay (ELISA).

The results of Morris Water Maze Test were shown in Table 1 and Table 2below.

TABLE 1 Results of Morris Water Maze Test on Aβ₁₋₄₀-induced AD rats fromday 1 to day 6 (n = 10) Arrival Time of Platform (Second) Groups Day 1Day 2 Day 3 Day 4 Day 5 Day 6 Control 68.75 ± 21.69 29.23 ± 14.28 16.96± 11.41 14.63 ± 7.17 17.58 ± 10.87 15.03 ± 7.69 Aβ 83.80 ± 19.65** 55.03± 22.13** 43.23 ± 31.39** 53.53 ± 22.13** 48.68 ± 22.95** 49.73 ±26.07** Aβ + 77.03 ± 29.92 39.10 ± 26.36^(##) 34.98 ± 23.42 31.10 ±23.26^(##) 30.00 ± 21.11^(##) 28.43 ± 23.45^(##) Donepezil Aβ + 74.98 ±27.89 40.45 ± 20.93^(##) 34.58 ± 24.64 29.73 ± 22.78^(##) 29.68 ±19.55^(##) 26.96 ± 22.35^(##) HEF-H Aβ + 79.65 ± 20.05 45.98 ± 21.6538.45 ± 20.46 38.80 ± 19.78^(##) 34.58 ± 21.83^(##) 35.23 ± 24.67^(#)HEF-M Aβ + 86.53 ± 27.08 50.95 ± 21.85 44.45 ± 31.45 48.13 ± 24.10 40.83± 19.91 39.23 ± 26.89 HEF-L *P < 0.05, **P < 0.01, Compared to controlgroup; ^(#)P < 0.05, ^(##)P < 0.01, Compared to Aβ group

TABLE 2 Frequencies of crossing platform location within two minutes onday 7 (n = 10) Group No. Control Aβ Aβ + Donepezil Aβ + HEF-H Aβ + HEF-MAβ + HEF-L 1 7 3 8 6 8 8 2 4 2 6 6 11 6 3 6 5 10 11 14 6 4 7 3 7 7 4 6 58 10 14 13 6 10 6 12 12 11 12 11 2 7 21 4 22 26 15 4 8 15 4 19 7 12 16 921 2 10 15 13 8 10  18 7 13 11 4 4 Mean ± SD 11.90 ± 6.44 5.20 ± 3.43**12.00 ± 5.16^(##) 11.40 ± 6.02^(#) 9.80 ± 4.05^(#) 7.00 ± 3.92 *P <0.05, **P < 0.01, Compared to control; ^(#)P < 0.05, ^(##)P < 0.01,Compared to Aβ

The Aβ group took a significantly longer time to arrive at the platformfrom day 1 to day 6 (Table 1), and showed lower frequencies in crossingthe platform location within 2 minutes on day 7 (Table 2) as comparedwith the control (p<0.01), which was correlated with the memoryimpairment in the rats of Aβ group due to the injection of Aβ₁₋₄₀.Comparing the treatment groups with Aβ group, the escape latencies ofAβ+D, Aβ+HEF-H, and Aβ+HEF-M groups were improved from day 1 to day 6,in particular, remarkably decreasing the time needed to arrive to theplatform from day 4 to day 6 (p<0.01) and increasing the frequency ofcrossing the platform location on day 7 (P<0.01 or P<0.05). Also,Aβ+HEF-L showed fewer escape latencies from day 4 to day 6 and higherfrequencies on day 7 compared to Aβ group.

These results demonstrated that the fat-soluble fraction of H. erinaceumhas the ability to improve recognition of space and memory in AD rats.

As shown in Table 3, after four weeks of treatment, the NGF content ofthe olfactory bulb, hippocampus, locus ceruleus, and cerebral cortex ofthe rats in all of treatment groups including Aβ+D, Aβ+HEF-H, Aβ+HEF-M,and Aβ+HEF-L had higher average values of NGF as compared to those incontrol and Aβ groups. In particular, the NGF content of the hippocampusand cerebral cortex in Aβ+D, Aβ+HEF-H, and Aβ+HEF-M groups showedsignificant increases (P<0.01 or P<0.05).

TABLE 3 NGF contents of cranial nerve nucleus of Aβ₁₋₄₀-induced AD rats(n = 10) NGF content (ng/g cranial nerve nucleus) Group Olfactory bulbHippocampus Locus ceruleus Cerebral cortex Control  9.20 ± 3.09 9.82 ±3.56 2.43 ± 0.64 5.08 ± 1.59 Aβ  9.89 ± 1.80 10.65 ± 2.33  2.46 ± 0.455.80 ± 0.98 Aβ+Donepezil 10.11 ± 2.10 14.20 ± 4.14* 3.07 ± 1.38  6.79 ±1.10* Aβ+HEF-H 10.89 ± 1.75 16.23 ± 6.16* 3.14 ± 0.95  7.59 ± 1.60**Aβ+HEF-M 10.72 ± 2.39 15.14 ± 5.92* 2.71 ± 0.76  6.69 ± 0.56* Aβ+HEF-L10.10 ± 1.91 11.54 ± 4.31  2.58 ± 0.85 5.90 ± 1.91 mean ± SD; n: Numberof rats; *P < 0.05, **P < 0.01, Compared to Aβ

Example 4

Recently, it has been reported that the neuronal cytotoxicity of amyloidbeta-peptide is very involved with oxidation stress and endoplasmicreticulum (ER) stress. Tunicamycin is a protein glycosylation inhibitorand generally used as an inducer of ER stress. This experiment wasconducted to investigate if HEF protects against ER-dependent neuro2acell death.

Neuro2a cells were cultured in a 96-well plate with Dulbecco's modifiedeagles medium at a cell density of 5,000 cells/well. After 1 day ofculture at 37° C., 0.5 μg/mL of tunicamycin (Wako, Japan) at varyingconcentrations (1 μg/mL or 10 μg/mL) of HEF was added to the medium. Thecells were incubated for 24 hours, and then the viability was measuredby 3-(4,5-dimethyl-2thiazolyl)2,5-diphenyl-2H-tetrazolium bromide (MTT)assay.

As shown in FIG. 3, the cell viability decreased to 11.6% after treatedwith tunicamycin, whereas the addition of 1 μg/mL and 10 μg/mL of HEFincreased the viability to 23.34% and 44.8% respectively. The resultsuggests that HEF may protect neuron from the cytotoxicity of amyloidbeta-peptide by reducing ER stress

Example 5

Five-week old male and female mice of the ICR strain were used in theexperiment. Five each of male and female mice were allocated to eachgroup, and the mice were not fed for about four hours prior to treatmentand then each was weighed. To the mice of the test group, HEF suspendedin saline was administered orally with a stomach tube at a dose of 300mg/kg. To the control group, the same volume of saline was administeredin the same manner as in the test group. Clinical observations were madeduring 14 days of the experiment period, and at the end of theexperimental period all mice were sacrificed for internal organexamination.

Throughout the experimental period, no animal death, no abnormalities ingeneral their general condition, and no significant difference in themean body weight were observed in either males or females after thetreatment. Also, no noteworthy changes were found in any organ of eithermales or females in any of the internal examinations. Consequently, itwas concluded that the LD50 of the HEF was higher than 300 mg/kg at asingle dose.

Example 6

Nine-week old male and female rats of the Sprague-Dawley strain wereused in the experiment. Ten each of male and female rats were allocatedto the control, HEF-H (high dose), HEF-M (middle dose), and HEF-L (lowdose) groups. To the rats of HEF-H, HEF-M, and HEF-L groups, HEFsuspended in 0.5% sodium carboxymethylcellulose (CMC-Na) wasadministered orally with a stomach tube separately at the doses of 120mg/kg, 60 mg/kg, and 7.2 mg/kg once per day and 6 times a week for 13weeks. To the control group, the same volume of 0.5% CMC-Na wasadministered in the same manner. Body weight was measured once a weekand the dose was adjusted based on the increase of body weight. Clinicalobservations were made during the 13 weeks of the experimental period,and at 24 hours after the end of the experiment period, each group wasdivided into two subgroups (five each of male and female). Blood wasdrawn from one subgroup for hematological and biochemical analyses, andthen these rats were sacrificed for internal organ examination. Themembers of the second subgroup were kept breeding without HEF for 4weeks of recovery period. At the end of the recovery period, blood wasdrawn for the hematological and biochemical analyses and all rats werethen sacrificed for internal organ examination.

Throughout the experimental and recovery periods, no animal death, noabnormalities in general condition, and no significant differences inthe mean body weight were observed in either males or females. Also, nonoteworthy changes were found in the hematological and biochemicalanalyses and in any organ of either males or females in any of theinternal examination.

EFFECT OF THE INVENTION

In the method of this invention, the process to obtain fat-solublecomponents by adding water into the concentrated ethanol extract is animportant improvement over the use of toxic organic solventfractionation used in the prior art. The method of this invention issafer and easier for use in industry compared to those described in theprior art and appears to possess a more stable yield rate.

The fat-soluble fraction obtained by the method disclosed above isdemonstrated to act not only as an inducer of NGF synthesis but also asan inhibitor of amyloid beta-peptide toxicity. Such both bioactivitieshave never been found simultaneously in any active compounds asdescribed in prior patents and publications.

The fat-soluble fraction of this invention has significant anti-dementiaeffects on AD rats when administered orally. Therefore, the fraction maybe useful for prevention and improvement of dementia, especiallyAlzheimer type dementia, and for improvement of memory loss and lack ofacuity.

The fraction of this invention shows no toxicities in single-dosetoxicity test and 90 days repeated dose toxicity test indicating it issafe for use in in vivo administration.

The fraction of this invention is able to be used as an activeingredient for pharmaceutical and/or health food products in tablet,capsule, granule, tincture, and beverage forms. For tablet, hardcapsule, and granule manufacturing, the fraction may be combined withexcipients such as, but not limited to, dicalcium phosphate, sucrosefatty acid ester, microcrystalline cellulose, lactose, silica or otherinactive fillers and binders well known to those skilled in the art. Forsoft capsules, the fraction may be combined with excipients, such as,but not limited to, vegetable oil, while in liquid form, carriersincluding, but not limited to, glycerin and ethanol may be used assolvent of the fraction. Methods of manufacturing all the above productconfigurations are known to those skilled in the art.

In addition, it should be noted that a variety of health food productscan be formulated by combining the fraction of this invention with otheractive ingredients such as, but not limited to Ginkgo, Docosahexaenoicacid (DHA), Phosphatidylserine, Choline as well as other medicinalmushrooms, herbs, vitamins, unsaturated fatty acids, phospholipids,choline, and minerals.

Further, the fraction of this invention that includes derivatives ofbenzyl alcohol, chromanes, and phosphatidylethanolamine can be used asan additive for health foods, general food products, and beverages. Thisfraction can be used not only for health foods or beverages designed forhumans but also for animal feed.

Thus it is seen that the objects of the invention are efficientlyobtained, although changes and modifications to the invention should bereadily apparent to those having ordinary skill in the art, whichchanges would not depart from the spirit and scope of the invention asclaimed.

1. A method for preparing a fat-soluble fraction of the fruiting body of Hericium erinaceum by the steps of: extracting the fruiting body of H. erinaceum with 95% ethanol at room temperature; separating said ethanol extract by filtration or centrifugation; concentrating said ethanol extract by removing ethanol under reduced pressure; adding 4˜8 volumes of water to said concentrated ethanol extract; after standing at 4˜10° C. for 8˜12 hours; collecting the resulting floating matter on the surface of said concentrated extract; and, drying said collected floating matter.
 2. The method for preparing a bioactive fraction as recited in claim 1 wherein said collection of the resulting floating matter is performed by skimming.
 3. The method for preparing a bioactive fraction as recited in claim 1 wherein said collection of the resulting floating matter is performed by pipetting.
 4. The method of preparing a bioactive fraction as recited in claim 1 wherein said fat-soluble fraction includes benzyl alcohol derivatives, chromane derivatives, and phosphatidylethanolamine derivatives.
 5. A bioactive fraction of H. erinaceum comprising fat-soluble components including benzyl alcohol derivatives, chromane derivatives, and phosphatidylethanolamine derivatives.
 6. The bioactive fraction as recited in claim 5 wherein said bioactive fraction includes anti-dementia and/or an anti-Alzheimer's compounds.
 7. The bioactive fraction as recited in claim 6 wherein said bioactive fraction is in the form of a pharmaceutical product.
 8. The bioactive fraction as recited in claim 6 wherein said bioactive fraction is in the form of a health food product.
 9. The bioactive fraction as recited in claim 6 wherein said bioactive fraction is in the form of a food product.
 10. The bioactive fraction as recited in claim 6 further comprising vitamins.
 11. The bioactive fraction as recited in claim 6 further comprising minerals.
 12. The bioactive fraction as recited in claim 6 further comprising herbs.
 13. The bioactive fraction as recited in claim 6 further comprising mushrooms.
 14. The bioactive fraction as recited in claim 6 further comprising unsaturated fatty acids.
 15. The bioactive fraction as recited in claim 6 further comprising phospholipids.
 16. The bioactive fraction as recited in claim 6 further comprising choline.
 17. The bioactive fraction as recited in claim 6 wherein said bioactive fraction is incorporated into a tablet.
 18. The bioactive fraction as recited in claim 6 wherein said bioactive fraction is incorporated into a capsule.
 19. The bioactive fraction as recited in claim 6 wherein said bioactive fraction is incorporated into a granule.
 20. The bioactive fraction as recited in claim 6 wherein said bioactive fraction is incorporated into a tincture.
 21. The bioactive fraction as recited in claim 6 wherein said bioactive fraction is incorporated into a beverage.
 22. A bioactive fraction of fat-soluble components of the fruiting body of H. erinaceum produced by the steps of: extracting the fruiting body of Hericium erinaceum with 95% ethanol at room temperature; separating said ethanol extract by filtration or centrifugation; concentrating the ethanol extract by removing ethanol under reduced pressure; adding 4˜8 volumes of water into the concentrated extract; standing at 4˜10° C. for 8˜12 hours; collecting the resulting floating matter on the surface of said concentrated extract; and, drying said collected floating matter.
 23. The bioactive fraction as recited in claim 22 wherein said collection of the resulting floating matter is performed by skimming.
 24. The bioactive fraction as recited in claim 22 wherein said collection of the resulting floating matter is performed by pipetting.
 25. The bioactive fraction as recited in claim 22 wherein said fat-soluble fraction includes benzyl alcohol derivatives, chromane derivatives, and phosphatidylethanolamine derivatives. 